Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model

AbstractLung cancer is a major public health problem in the western world, and gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumour tissue. Some of the main factors inhibiting systemic delivery are found in the blood stream in the form of red and white blood cells (WBCs) and serum components. Mesenchymal stem cells (MSCs) have been shown to home to tumour sites and could potentially act as a shield and vehicle for a tumouricidal gene therapy vector. Here, we describe the ability of an adenoviral vector expressing TRAIL (Ad.TR) to transduce MSCs and show the apoptosis‐inducing activity of these TRAIL‐carrying MSCs on A549 lung carcinoma cells. Intriguingly, using MSCs transduced with Ad.enhanced‐green‐fluorescent‐protein (EGFP) we could show transfer of viral DNA to cocultured A549 cells resulting in transgenic protein production in these cells, which was not inhibited by exposure of MSCs to human serum containing high levels of adenovirus neutralizing antibodies. Furthermore, Ad.TR‐transduced MSCs were shown not to induce T‐cell proliferation, which may have resulted in cytotoxic T‐cell‐mediated apoptosis induction in the Ad.TR‐transduced MSCs. Apoptosis was also induced in A549 cells by Ad.TR‐transduced MSCs in the presence of physiological concentrations of WBC, erythrocytes and sera from human donors that inhibit or neutralize adenovirus alone. Moreover, we could show tumour growth reduction with TRAIL‐loaded MSCs in an A549 xenograft mouse model. This is the first study that demonstrates the potential therapeutic utility of Ad.TR‐transduced MSCs in cancer cells and the stability of this vector in the context of the blood environment.</jats:p

Caspase-10: a molecular switch from cell-autonomous apoptosis to communal cell death in response to chemotherapeutic drug treatment.

The mechanisms of how chemotherapeutic drugs lead to cell cycle checkpoint regulation and DNA damage repair are well understood, but how such signals are transmitted to the cellular apoptosis machinery is less clear. We identified a novel apoptosis-inducing complex, we termed FADDosome, which is driven by ATR-dependent caspase-10 upregulation. During FADDosome-induced apoptosis, cFLIPL is ubiquitinated by TRAF2, leading to its degradation and subsequent FADD-dependent caspase-8 activation. Cancer cells lacking caspase-10, TRAF2 or ATR switch from this cell-autonomous suicide to a more effective, autocrine/paracrine mode of apoptosis initiated by a different complex, the FLIPosome. It leads to processing of cFLIPL to cFLIPp43, TNF-α production and consequently, contrary to the FADDosome, p53-independent apoptosis. Thus, targeting the molecular levers that switch between these mechanisms can increase efficacy of treatment and overcome resistance in cancer cells.Cell Death and Differentiation advance online publication, 3 November 2017; doi:10.1038/cdd.2017.164

The Molecular Mechanisms of 5-Fluorouracil-Induced Apoptosis in Human Colorectal Cancer Cells and the Use of 5-FU as a Sensitising Agent to TRAIL

Colorectal cancer is the third most common cause of death worldwide and despite many advances in recent years resistance to apoptosis induced by chemotherapeutic drugs, such as 5-fluorouracil (5-FU), remains a serious issue. A better understanding of how chemotherapeutic drugs induce apoptosis in colon cancer cells will better inform us on how to develop novel therapeutic strategies to overcome this resistance. We investigated cell death induced by 5-FU in the colon cancer cell line HCT116, and found that not only cell death, but also cytochrome-c release was dependent on an upstream caspase activity. Upon further investigation, we discovered a large (1-2 MDa) apoptosis-inducing complex forming following 5-FU-mediated RNA stress. This complex could be purified and identified by sucrose gradient density fractionation and was found to contain the core components caspase-8, FADD and RIP1 with interaction from Bid. In the absence of caspase-8 and FADD, complex formation and apoptosis was abolished. This complex forms without involvement of death receptors such as CD95 or the TRAIL receptors DR4 and DR5 despite a clear upregulation of DR5 protein levels following 5-FU. Futhermore, knocking down DR5 had no effect on initial caspase-8 cleavage and did not prevent complex formation. This complex forms upstream of the mitochondria, evidenced by overexpression of Bcl-2 or knocking down Bax or Bid. In addition we could demonstrate the inducible interaction between the complex members caspase-8 and FADD as well as FADD with RIP1 by co-immunoprecipitation, and the inducible interaction of FADD molecules following 5-FU stimulation. We also explored the contribution of the 5-FU-induced DR5 upregulation to Tumour necrosis-factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis in HCT116 cells. TRAIL selectively induces rapid apoptosis in most tumour cell types, and represents a promising anti-cancer agent. Subtoxic doses of TRAIL-induced apoptosis could be enhanced by co-treatment with low doses of 5-FU in a caspase dependent manner. Sensitisation to TRAIL involved enhanced DR5 expression and activation of the caspase cascade. Our results demonstrate that caspase-8 expression is necessary for this 5-FU-mediated TRAIL sensitivity. Finally, we demonstrated that low-dose 5-FU pre-treatment sensitises HCT116 cells to Mesenchymal stem cellmediated delivery of sTRAIL in vitro

IFN-γ combined with targeting of XIAP leads to increased apoptosis-sensitisation of TRAIL resistant pancreatic carcinoma cells

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a specific and potent inducer of apoptosis in cancer cells, but the resistance of many tumour cells to TRAIL still represents a major hurdle for the clinical treatment of tumours with TRAIL. As apoptosis is regulated by the balance of activities of several anti-apoptotic factors and pro-apoptotic factors, we analysed the relative contribution of the two sides and found that down-regulation of Bcl-x L and in particular XIAP, but not c-Flip, sensitised the TRAIL resistant pancreatic cancer cell line Panc-1. A combination of both XIAP and Bcl-x L knock-downs showed no substantial added benefit indicating that both act in the same pathway. Notably, the degree of sensitisation by silencing of anti-apoptotic genes was further elevated by concomitantly increasing the pro-apoptotic potential in Panc-1 cells through over-expression of TRAIL-R1 or IFN-γ-mediated increases in caspase-8 levels. Similar sensitisation effects were obtained for another TRAIL-resistant pancreatic tumour cell line, AsPC-1. Our findings demonstrate that modulation of the balance between anti- and pro-apoptotic pathways from both sides by inhibition of apoptosis-antagonists and stimulation of pro-apoptotic factors provides the best way to enhance the anti-tumourigenic effect of TRAIL. © 2011 Elsevier Ireland Ltd

Mesenchymal stem cells expressing trail lead to tumour growth inhibition in an experimental lung cancer model

Lung cancer is a major public health problem in the western world, and gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumour tissue. Some of the main factors inhibiting systemic delivery are found in the blood stream in the form of red and white blood cells (WBCs) and serum components. Mesenchymal stem cells (MSCs) have been shown to home to tumour sites and could potentially act as a shield and vehicle for a tumouricidal gene therapy vector. Here, we describe the ability of an adenoviral vector expressing TRAIL (Ad.TR) to transduce MSCs and show the apoptosis-inducing activity of these TRAIL-carrying MSCs on A549 lung carcinoma cells. Intriguingly, using MSCs transduced with Ad.enhanced-green-fluorescent-protein (EGFP) we could show transfer of viral DNA to cocultured A549 cells resulting in transgenic protein production in these cells, which was not inhibited by exposure of MSCs to human serum containing high levels of adenovirus neutralizing antibodies. Furthermore, Ad.TR-transduced MSCs were shown not to induce T-cell proliferation, which may have resulted in cytotoxic T-cell-mediated apoptosis induction in the Ad.TR-transduced MSCs. Apoptosis was also induced in A549 cells by Ad.TR-transduced MSCs in the presence of physiological concentrations of WBC, erythrocytes and sera from human donors that inhibit or neutralize adenovirus alone. Moreover, we could show tumour growth reduction with TRAIL-loaded MSCs in an A549 xenograft mouse model. This is the first study that demonstrates the potential therapeutic utility of Ad.TR-transduced MSCs in cancer cells and the stability of this vector in the context of the blood environment